Factor Xa is the serine protease component of the enzymatic complex termed prothrombinase, the only known physiological activator of prothrombin. The broad long-term objective of this work is to identify structural determinants on FXa that are important for its function within the prothrombinase complex. This proposal contains two specific aims. In the first aim, we will determine whether surface exposed regions in the catalytic domain of FXa define cofactor and substrate exosites. The hypotheses from this aim to be tested are, 1) the 162 helix on FXa (residues 162-170, chymotrypsin numbering) provides a critical binding surface for FVa, and 2) the negatively charged 39-loop on FXa (residues 35-41) makes specific electrostatic contacts with its substrate prothrombin. In the second aim, we will determine whether sodium binding to the 225-loop (residues 220-226) on FXa modulates its activity. It is our hypothesis that in the absence of sodium binding the ability of FXa to recognize macromolecular substrates is altered. To carry these aims out, we will 1) prepare recombinant variants of FXa using site-directed mutagenesis, 2) produce the variants in a mammalian expression system, and 3) characterize the proteins with respect to: purity, gamma-carboxyglutamic acid content, activity in clotting assays, cleavage of chromogenic and macromolecular substrates, and interactions with non-protein and protein cofactors. Choice of mutations will be based on the available crystal structure of FXa, and on previous experimental findings defining molecular determinants in homologous enzymatic complexes. Understanding and identifying the complex molecular interactions involved in the assembly and function of prothrombinase, is a key first step in the development of pharmacologic agents aimed at controlling local concentrations of thrombin. In addition, this proposal will contribute to our understanding of the determinants of FXa's substrate specificity, which may ultimately lead to new strategies for modulating the catalytic properties of this critical serine protease.